Comparative Assessment of Three Medicinal Plants against Diabetes and Oxidative Stress Using Experimental and Computational Approaches

The hilly and rural areas' people of Bangladesh have a great history of putting into use numerous traditional medicinal plants to cure diseases. Therefore, with ethanol extract of Molineria capitulata (EEMC), methanol extract of Trichosanthes tricuspidata (METT), and methanol extract of Amorphophallus campanulatus (MEAC), we mandate evaluation of in vitro α-amylase inhibition, antioxidants, and molecular docking, and ADMET/T analysis. According to iodine starch methods, α-amylase inhibition was performed, and quantitative total phenolic and flavonoid content was determined by established methods, whereas DPPH free radical scavenging and reducing power assays were performed in previously established protocols, respectively. A comparative study among three plants (EEMC, METT, and MEAC) possessed a significant (p < 0.01) effect but EEMC showed the highest impact on enzyme inhibition. Plants in the measuring phenolic content METT and flavonoid measurement MEAC displayed most potent in the same way in the DPPH test was METT, and in reducing power capability MEAC has showed the highest effect between three extracts. Docking's study also reveals the compounds of METT (Cyclotricuspidoside A and Cyclotricuspidoside C) exhibit the superior score among all the compounds. This finding indicates that EEMC, METT, and MEAC substantially impact α-amylase inhibition along with antioxidants. In silico study also reveals the potency of these plants, but further in-depth, precise molecular studies are needed.


Introduction
Diabetes has been afecting countless people over the world, which is due to starch, fat, and protein metabolic upset [1]. Progressing estimations exhibit that nearly 2.8% of the total population has diabetes and this number will reach 5.4% by 2025 [2]. We may face a worldwide type 2 diabetes disorder pandemic within the following 20 years. Even though new diabetic disorders the entirety relies on the glucose models used to symbolize diabetes, the rate and predominance of type 2 diabetes have been expanding [3]. Diabetics additionally seem to build the creation of professional provocative cytokines and incendiary arbiters, for example, interleukin-1 (IL-1), interleukin-6 (IL-6), tumor rot factor-α(TNF-α), macrophage chemoprotectant-1 (MCP-1), and nitric oxide (NO) which are likewise connected to the pathogenesis of diabetes [4]. Although some salve has been used extensively during the past few decades generally, the truth is that they have also been refecting unforeseen scenarios. Terefore, keeping up stable and lower blood glucose can accomplish by postponing glucose assimilation through restraint of sugar hydrolyzing proteins, for example, α-glucosidase and α-amylase in the stomach-related tract [5]. Te α-amylase (α-1,4-glucan-4glucanohydrolases) is an eminent secretory result of the pancreas and salivary organ liable for the underlying advance in the hydrolysis of complex sugar to a blend of oligosaccharides and disaccharides in the intestinal mucosa [6]. Tere are a few points of interest in common home-grown medications, for example, a decrease in the danger of reactions, the viability of interminable conditions, far-reaching accessibility, and minimal efort. Consequently, inhibitors of the α-amylase compound, which is separated from plants, could be developing contenders to control hyperglycemia in diabetic patients [7].
Reactive oxygen species (ROS) contain highly reactive molecules utilizing oxygen metabolism [8,9]. ROS, such as hydroxyl radicals, superoxide radicals, peroxyl radicals, and hydrogen peroxide are constantly generated as byproducts of metabolic reactions or from several exogenous factors. Tey serve an important physiological function in low to moderate concentrations, such as immunocompetence, apoptosis, hormonal regulation, signal transduction, transcription factors, and adaptive responses to enzymes [4]. But an excessive production of ROS and a weakened antioxidant defense system often lead to the development of oxidative stress (OS) [5,6]. Oxidative stress (OS) is one of the key factors in inducting a variety of chronic and degenerative diseases, including atherosclerosis, ischemic heart attack, aging, and diabetes mellitus; along with this, cancer, immunosuppression, and neurological disorder [7]. Natural antioxidants obtained from plant sources are considered a signifcant approach in retarding the prognosis of diabetes and other chronic diseases as they are capable of neutralizing ROS thus alleviating oxidative stress [8][9][10]. Secondary plant metabolites such as favonoids and tannins are rich in antioxidant activity, which are believed to be efcient in resisting the destruction of pancreatic β-cells and diabetesinduced ROS production. Tus, a plant having a strong enzyme inhibitory and antioxidant potential may be considered an important therapeutic candidate for managing diabetes [11].
Molineria capitulata is generally a stemless, stout herbtype plant known as palm grass, which is up to 1 m in length and belongs to Hypoxidaceae [12,13]. Traditionally diferent parts of M. capitulata are used for various purposes such as rhizomes decocted with herbal medicines for the management of consumptive cough, asthenia, impotence, and spermatorrhea. In India, it was initially recorded as a treatment for hemorrhoids, asthma, jaundice diarrhea, colic, gonorrhea, and roots and leaves used in country liquor. Te contemporary investigation addressed the presence of several isolated phytoconstituents such as Crassifoside A, Breviscaside A, Crassifogenin C, Crassifoside D, Curcapital, Isocurculigine [14]. In Addition, the current study proposed the hypoglycemic and anthelminthic activities proclaimed from roots [15].
Amorphophallus campanulatus is commonly known as Elephant foot yam. Generally, it is a herbaceous, longstanding plant, and it can be 0.75 m in height and belongs to the family of Arecaceae [20]. Traditionally, it has been used for several purposes for example tumors, spleen enlargements, asthma, and rheumatism. Te plant's tuberous roots have also been found to have tonic, stomach, and appetizing properties. Infusion of leaf stalks is useful in bites of poisonous insects [21]. Te recent study evaluated phytoconstituents such as Stigmasterol; β-Sitosterol; Campesterol; 1,3,5-Benzenetriol; Vitamin E acetate; and Squalene. Te latest study reported anthelminthic activity from tuber [22].
Although these three plants have several important traditional uses, no scientifc research has been carried out to determine their activity against diabetes and oxidative stress; that is why the present study has aimed to evaluate those three medicinal plants' antidiabetic and antioxidant activity through experimental (in vitro study) and computational techniques (molecular docking, ADME/T study, and PASS predictions).  Evidence-Based Complementary and Alternative Medicine

Preparation of Plant Material and Extract Preparation.
Normally each of the plant materials was collected in fresh condition. Ten, the leaves dried under shade and ground for 10 days. Te materials were ground to obtain coarse powder and fnally preserved in an airtight container. Te leaves powder (100 gm) was soaked in 500 ml of ethanol (M. capitulata) and methanol (A. campanulatus and T. tricuspidata) for 7 days at room temperature (25.0 ± 0.5)°C. Ten, the solvent was refned and evaporated extra liquid portion through water bath to leave a viscous mass. Furthermore, it is placed at room temperature for a while for getting dried extract.

In Vitro Study: In Vitro α-Amylase Inhibition Assay for
Antidiabetic Activity. Te assay was acted in an act following based on the starch-iodine test [23]. In brief, 1 ml of plant extract of diferent concentrations (1000−125 μg/ml) was added to 1 ml of Na₃PO₄ bufer (pH 6.9 full of 6 mmol NaCl) containing 0.04 units of α-amylase solution. Te mixture was incubated at 37°C for 10 min to complete the reaction. Ten, 1 ml soluble starch (1% w/v) was added to each concentration and again incubated at 37°C for 15 min. Again 1 M HCl (40 μL) was added, then followed by the addition of 200 μl of iodine reagent. Te absorbance was estimated at 620 nm in a UV-spectrophotometer.

Qualitative Estimation of 2,2-Diphenyl-l-Picryl-
Hydrazyl-Hydrate (DPPH). Te free radical scavenging performance of samples was carried out in terms of hydrogen donating or radical-scavenging ability using the stable radical DPPH [24]. In short, a test sample or standard 0.1 ml at diferent concentrations (500 to 15.625 μg/ml) was added to 3 ml of a 0.004% methanol solution of DPPH in a test tube. Te mixture was incubated for 30 min at room temperature to complete the reaction. Ten, the absorbance was measured at 517 nm by using a UV-visible spectrophotometer against a blank. Te % inhibition was carried out by the following equation ((A0 − A1)/A0) × 100, where A0 denotes the absorbance of the control and A1 denotes the test sample or standard absorption.

Qualitative Estimation of Reducing Power.
Te transformation of Fe 3+ to Fe 2+ can be visualized by the determination of reducing power [25]. In brief, 1 ml of the test sample or standard (500 to 15.625 μg/ml) was blended with 0.2 M phosphate bufer 2.5 ml (pH 6.6) along with potassium ferricyanide 2.5 ml, (1% w/v). Te blend was incubated to complete the reaction at 50°C and the duration was 20 min. Furthermore, 2.5 ml of trichloroacetic acid (TCA) (10% v/v) was added to the mixture and then centrifuged for 10 min at 3000 rpm. An equal amount (2.5 ml) supernatant layer of the solution and distilled water was mixed after that 0.5 ml of FeC1 3 (0.1% w/v) was added. Ten, the absorbance was evaluated at 700 nm by using a UV-visible spectrophotometer against blank.

Quantitative Estimation of TPC.
Applying the Folin-Ciocalteu reagent to the mixture, the quantity of TPC was carried out [26]. In brief, 0.5 ml of standard/test sample (1.00 mg/ml) at diferent concentrations (500 to 15.625 μg/ ml), 2.5 ml of the Folin-Ciocalteu reagent (FCR) was added.
After that within 0.5 to 8 min, 2 ml of Na 2 CO 3 (7.5%) was added. Te mixture was incubated for 5 min to carry out the reaction at 50°C and then cooled. Ten, the absorbance was measured at 760 nm by utilizing the UV-visible spectrophotometer opposite to the blank.

Quantitative Estimation of TFC.
By executing an aluminum colorimetric assay, the quantity of TFC was evaluated [26]. About 1.00 ml of test/standard (100 to 12.5 μg/ml) was blended with 3.00 ml of methanol (CH 3 OH), 0.2 ml of 10% AlCl 3 , 0.2 ml of 1 M potassium acetate, and 5.6 ml of distilled water. Te mixture was incubated at room temperature (25°C) to carry out the reaction for half an hour. Ten, the absorbance was estimated at 420 nm with the help of a UV-visible spectrophotometer versus blank.

Chemical Compounds and Proteins.
Selective compounds were downloaded from PubChem [27] as a 2D SDF fle for comparative docking investigation against a standard candidate. Te compounds were ascorbic acid, gallic acid, and acarbose, selected from the previous investigation [14,18,22]. Ten, relevant proteins were taken as PDB fles from Protein Data Bank [28]; these proteins were pancreatic α-amylase [PDB: 3BAJ] and uricase [PDB: 1R4U].

Ligand and Protein Preparation.
Te selected compounds and standard drugs were prepared to utilize LigPrep wizard, a bioinformatics tool included in Maestro (Schrödinger software v 11.1). Te compounds were fxed as project-table fles; in addition, the other parameters were kept in the default set-up. Tereafter, importing the anticipated protein as PDB-format as well as performing preprocess job by dint of Protein Preparation Wizard. Subsequently, the protein molecules were prepared by eliminating the water molecules (<3 H-bonds to nonwaters). Te force feld is fxed at OPLS3 during the minimization process. Furthermore, supplementary parameters were kept in the default situation. Afterward, the receptor grid was generated by the use of the Receptor-Grid Generation Wizard. PockDrug [28], an online tool, was used to pick the best docking goal in keeping with the highest druggability probability value. Te X, Y, and Z axis has been kept within 6 to 14 in case of the advanced setting of a target site.

Glide Standard Precision (SP) Ligand Docking.
Te fexible-docking was directed between protein molecules and legends to recognize possible biological mechanisms completed by Maestro (v 11.1). Te docking interaction was executed utilizing the Ligand-Docking Wizard to promote Evidence-Based Complementary and Alternative Medicine the ligands for docking based on binding strength. Troughout this operation, all factors were preserved in the default function, and to get the best output, the docking job was being executed for several times. Lastly, a docking spreadsheet was gathered for additional data analysis. For a better understanding of the docking relations, 2D as well as 3D fgures were occupied by a molecular imagining tool (Discovery studio-v 4.1).

ADME, Toxicological Property, and PASS Prediction
Analysis. Diverse biokinetics properties in addition to toxicological properties such as absorption, distribution, metabolism, excretion, and toxicity (ADME/T) are measured during drug development. Te ADME and toxicological properties defned above are estimated by [29] and Admet-SAR [30], respectively, which are online bioinformatics depositories. Depending on Lipinski's and Veber's rules, subsequent parameters were audited to guess drug-likeness behaviors, for instance, molecular weight (MW), hydrogenbond acceptor (HBA), hydrogen-bond donor (HBD), lipophilicity (logP), number of the rotatable-bond (NRB), and topological polar surface area (TPSA). Ten, again rat-acute toxicity, acute-oral toxicity, as-toxicity, and carcinogenic were measured through assessment of toxicity. PASS Online [31], an online bioinformatics platform, was used to accomplish biological prediction.

In Vitro α-Amylase Inhibition Assay for Antidiabetic
Activity. In this α-amylase inhibition, assay all three plant extracts demonstrated promising antidiabetic bioactivity comparison with acarbose as a potential antidiabetic drug described in Table 1 and Figure 1. Te percentage of α-amylase inhibition increased with the increase of concentration density. Between 150 and 875 μg/mL concentrations, the EEMC showed the highest inhibition rate of the remaining two samples (METT and MEAC) with the IC 50 value of 300.9 ± 3.38 μg/mL. However, the IC 50 value of the α-amylase inhibition assay was 133.3 ± 0.82 μg/mL. Te order for percentage of α-amylase inhibition was as follows: acarbose > EEMC > MEAC > METT at 1000 μg/mL conc.

Quantitative Estimation of DPPH.
Utilizing the DPPH free radical scavenging qualitative assay, the antioxidant activity of EEMC, METT, and MEAC was studied compared to the standard candidate (ascorbic acid) summarized in Table 2 and Figure 2. Among all three samples extracted, the METTsignifcantly showed the highest scavenging activity at 500 μg/mL concentration (P < 0.01) with concentrationdependent tendency, and the minimum IC 50 value was 81.88 ± 0.99 μg/mL while the IC 50 value was 116.7 ± 2.21 and 162 ± 1.7 μg/mL for EEMC and MEAC, respectively. However, the minimum inhibitory concentration value of the standard drug was 45.43 ± 0.75 μg/mL.

Quantitative Estimation Reducing Power Activity.
By maintaining a concentration-dependent manner EEMC, METT, and MEAC moderately showed a reducing power efect compared to ascorbic acid at 700 nm absorbance represented in Figure 3. Te MEAC showed maximum absorbance of 0.840 at 500 μg/mL conc. while ascorbic acid showed 2.587 at the same concentration. Te sequence for reducing power activity was as followed: ascorbic acid > MEAC > EEMC > METT at 500 μg/mL concentration.

Quantitative Estimation of TPC and TFC.
Te quantitative investigation of antioxidant-related phytochemicals with TPC as well as TFC of EEMC, METT, and MEAC are summarized in Table 3. Te METT exhibited the highest total phenol content (107.50 ± 1.58 mg GAE/g), whereas EEMC and METT showed 57.92 ± 1.72 and 65.78 ± 1.06 mg GAE/g phenolic contents, respectively. Ten, again EEMC exhibited the highest total favonoid            (Table 7).

Pass Predictions.
Pass investigation demonstrated hypothetical pharmacological action of each of the main compounds within MEAC, METT, and MEAC. We     appraised diferent organic activities for each compound and considered the values of Pa > Pi and Pa > 7. Tis assumption proposes several immense compounds studied, such as antidiabetic, antioxidant, insulin promoter, free radical scavenger, and α-glucosidase inhibitor activities which are related to our present study. Te guessed pharmacological activities of all (6 compounds of each plant) main compounds are presented in Tables 8  and 9.

Discussion
Currently, wild plants are regarded to have high dietary values because of superior fber, polyphenol substances, and higher antioxidant capacity than cultured plants. Furthermore, numerous plants have exhibited to be fantastic efcacy in continual diseases for example cardiovascular diseases and diabetes. Quite a bit of this information has been orally passed from age-to-age which has prompted the advancement of the typical health care system, practiced in diferent nations of the world [32,33]. Instinctively, inhibitors of α-amylase by food-grade natural sources deliver an appealing remedial way to deal with the treatment of postprandial hyperglycemia through diminishing glucose discharge from starch, which might be possibly valuable in the treatment of diabetes mellitus and weight problems [8]. On the other hand, the cell's protection components can be either endogenous or exogenous; for this, the indispensable section is antioxidant [34]. In this study, plotted three individual (EEMC, MEAC, and METT) three plant extracts for in vitro inhibition of α-amylase and antioxidant activities along with in silico study (molecular docking, ADME/T, PASS predictions, and DFT activities) have been exhibited.
In the present study, inhibition of α-amylase of three plants (EEMC, MEAC, and METT) explores the signifcant efect, but EEMC demonstrates the highest impact among the other three extracts as compared to a reference to the standard drug (acarbose) additionally which also is statistically signifcant. Tis inhibition frmly demonstrated the presence of some phytoconstituents; these constituents are responsible for this inhibition and may occur following compounds that are responsible for these activities such as saponin, steroid, and terpenoid [35]. Contemporary investigations have stated the enzyme inhibitory activities of plant phenolics with a solid inhibitory impact on α-glucosidase, but a gentle impact on α-amylase, therefore proposing its utilization for the cure and the executives of diabetes [36]. Te α-amylase and α-glucosidase are alluded to as promising therapeutic efect in diabetes which inhibits and delay the action of starch consumption enzymes [37].
Moreover, the antioxidant efcacy of separate three plant extracts (EEMC, MEAC, and METT) was revealed based on the qualitative DPPH free radical scavenging activity and reducing power capacity assay and quantitative total TPC and TFC. Furthermore, this investigation showed among three diferent plants extract; METT possesses a potent antioxidant efect as compared to thestandard (ascorbic acid). Te antioxidant mechanism privileges the reduction formation of the hydroxyl radicals throughout lipid peroxidation. Te transition metallic ion Fe 2+ has the potential to uproot a single electron by way of the distinctive feature of which it may disable the placing and extension of numerous radical responses [38]. Apart from this, the study also reveals reducing power activity of EEMC, MEAC, and METT has increased in a concentration-dependent manner. Te presence of polyphenolic compounds (favonoids, phenolic acids, and tannins) may be responsible for reducing the  power activity of plants which also indicates the strong potentiality of the antioxidant activity [39]. Furthermore, molecular docking is one of the incredible assets to exploring the dynamic site of the protein and additionally comprehending and clarifying the binding associations among the ligands and desired protein [40]. In the molecular study of α-amylase inhibition study, we have selected 18 (each plant represents the 6 compounds) major compounds of EEMC, METT, and MEAC. We interact compounds individually with the targeted protein pancreatic α-amylase [PDB: 3BAJ]; in a comparison study, we noticed that the compounds of METT (Cyclotricuspidoside A and Cyclotricuspidoside C) had displayed the highest docking score, which is almost closed to the standard reference drug acarbose. Along with this, the Crassifogenin C and Breviscaside A compounds of METT showed the highest docking score than the Stigmasterol and β-Sitosterol compounds of MEAC. Docking score, Glide Emodel, and Glide energy was considered.
Subsequently, in silico antioxidant molecular docking study of 18 compounds of EEMC, METT, and MEAC (each plant of 6 compounds) was carried out through interaction with the targeted protein Uricase [PDB: 1R4U]. In this study, we evaluated the compounds of METT (Cyclotricuspidoside A and Cyclotricuspidoside C) exhibited prominent results as compared to other compounds which are higher than the standard, on the other hand EEMC (Isocurculigine and Crassifoside A) and MEAC (1,3,5-Benzenetriol and Campesterol) also possess potent outcome as compared to the standard which is also most closed to the standard. However, we considered the Docking Score, Glide Emodel, and Glide Energy. It could have the function of rival antioxidant efect on the protein, those facts are in complete agreement with the associated good docking rating and binding afnity.
Furthermore, for 18 compounds, we established their pharmacokinetic properties' physiochemical aspects, and drug-likeness, through ADME analysis, which is an online server basis program. We follow the two rules, one is Lipinski's rule and the other is Veber's rule; according to these rules, Cyclotricuspidoside A and Cyclotricuspidoside C of METT were violets maximum rules; on the other hand, those two compounds did not obey Veber's rules. It is proclaimed that as much as lower molecular weight, higher tendency to dissolving, and the ability of hydrogen bonds to have high permeation ability with favorable absorption rate and bioavailability.
Subsequently, we performed toxicity tests online to fnd the toxicity properties of EEMC, METT, and MEAC; we considered a few parameters such as Ames toxicity, carcinogens, and acute oral toxicity. We also noticed the LD50, but among the 18 compounds from three plants, only two compounds (Cyclotricuspidoside A and Cyclotricuspidoside C) possessed the highest LD50 value.
Moreover, for the prediction of efcacy of the plant substance activity, we analyzed by prediction of activity spectra for substances (PASS), which assessed the biological activity of prediction. Te outcome proposes many activities, among them we ascertained 18 compounds of EEMC, METT, and MEAC possible activity values, which lays under the Pa range 0.123 to 0.780.

Conclusions
Te output may indicate that the EEMC, METT, and MEAC possess profound α-amylase inhibition and antioxidant activities. Terefore, the present study proposes a scientifc basis for implementing this plant to manage various illnesses. However, this is only an initial study. Further indepth, precise molecular studies are warranted in in silico analysis to reveal that these compounds will be the source of the new biological activity.

Data Availability
All data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.